Mutant glucose dehydrogenase

ABSTRACT

A mutant glucose dehydrogenase having an improved substrate specificity to glucose, which is a protein comprising the amino acid sequence depicted in SEQ ID NO:3 or an amino acid sequence having the substitution, deletion, insertion or addition of one or more amino acid residues excluding amino acid 365 in the amino acid sequence depicted in SEQ ID NO:3 and having a glucose dehydrogenase activity, the amino acid residue corresponding to amino acid 365 being substituted by other amino acid residue.

TECHNICAL FIELD

The present invention relates to a mutant glucose dehydrogenase showingimproved substrate specificity. The mutant glucose dehydrogenase of thepresent invention can be suitably used for glucose sensors, glucoseassay kits and so forth, and is useful in the fields of biochemistry,clinical medicine, and so forth.

BACKGROUND ART

In recent years, a variety of enzymes are used as biosensor elements.Glucose oxidases (GODs) have already been practically used as sensorelements for measuring blood glucose levels for the purpose of diagnosisof diabetes. However, GODs suffer from a problem that they are affectedby dissolved oxygen in samples. Therefore, glucose dehydrogenases(GDHs), which are not affected by dissolved oxygen in samples, aredrawing attentions as alternatives of GODs.

As GDHs, one requiring NAD(P)⁺ as a coenzyme (E.C.1.1.1.47), onerequiring pyroloquinoline quinone (PQQ) as a coenzyme (PQQGDH;E.C.1.1.99.17) etc. have been reported. GDH requiring NAD(P)⁺ as acoenzyme suffers from a problem as a sensor element that NAD(P)⁺ needsto be added to the assay system. On the other hand, it is unnecessaryfor coenzyme-binding type GDHs such as PQQGDH to add a coenzyme to theassay system.

Further, sensor elements are desired to exhibit a stability that thefunction as a sensor is not lost even when they are continuously used orleft at room temperature.

Since enzymes derived from thermophilic bacteria which grow at hightemperature generally exhibit high thermostability, and high stabilityeven in long-term storage, continuous use and so forth, application ofthem as sensor elements is expected. However, although GDHs derived fromThermoplasma acidophilum and Sulfolobus solfataricus have been reportedas thermostable GDHs derived from thermophilic bacteria, both of themrequire NAD(P) e as a coenzyme.

On the other hand, thermostable GDH produced by Burkholderia cepacia, amoderately thermophilic bacterium, is an FAD-binding type GDH, and theenzymological characteristics thereof such as optimum reactiontemperature, thermostability and substrate specificity have already beenelucidated (Patent document 1). This GDH usually exists as aheterooligomer consisting of a catalytic subunit (α-subunit) showinghigh heat resistance, an electron transfer subunit (β-subunit), which iscytochrome C, and γ-subunit of which function is unknown, and itsoptimum reaction temperature is 45° C. These subunits are dissociated bya heat treatment at a temperature higher than 50° C. to release theα-subunit monomer of which optimum reaction temperature is 75° C. Theα-subunit monomer is thermostable and exhibits 80% or more of residualactivity even after a heat treatment at 60° C. for 30 minutes. The genescoding for these subunits have also already been isolated (Patentdocuments 1 and 2).

However, coenzyme-binding type GDHs generally exhibit a broad substratespecificity, and also react with maltose, galactose and so forth inaddition to glucose. When they are applied as a glucose sensor formonitoring blood sugar levels of diabetic patients, and the diabeticpatients have such severe symptoms that peritoneal dialysis must beperformed, there is a risk that values higher than the true blood sugarlevels may be obtained, because a large amount of maltose is containedin the dialysate. GDH derived from Burkholderia cepacia also exhibitsreactivity to maltose and galactose in addition to glucose.

A technique of changing substrate specificity of GDH by introducing anamino acid substitution mutation is known. As such mutant GDHs, forexample, there are known PQQGDHs derived from E. coli (Patent documents3 and 4), Acinetobacter calcoaceticus (Gluconobacter calcoaceticus)(Patent document 5), and Acinetobacter baumannii (Patent documents 6 to8) requiring pyroloquinoline quinone as a coenzyme.

[Patent document 1] U.S. Patent Application No. 2004/0023330

[Patent document 2] International Patent Publication WO03/091430

[Patent document 3] Japanese Patent Laid-open (Kokai) No. 10-243786

[Patent document 4] Japanese Patent Laid-open No. 2001-197888

[Patent document 5] Japanese Patent Laid-open No. 2004-173538

[Patent document 6] Japanese Patent Laid-open No. 2004-313172

[Patent document 7] Japanese Patent Laid-open No. 2004-313180

[Patent document 8] Japanese Patent Laid-open No. 2004-344145

DISCLOSURE OF THE INVENTION

An object of the present invention is to provide an FAD-binding type GDHshowing an improved substrate specificity to glucose.

The inventors of the present invention conducted various researches inorder to achieve the foregoing object. As a result, they found that bymodifying the amino acid sequence of the FAD-binding type GDH derivedfrom Burkholderia cepacia at a specific site, the reactivity thereof tosugars other than glucose could be decreased while maintaining thereactivity to glucose, and thus accomplished the present invention.

That is, the present invention provides the followings.

(1) A mutant glucose dehydrogenase having the amino acid sequence of SEQID NO: 3 or an amino acid sequence of SEQ ID NO: 3 includingsubstitution, deletion, insertion or addition of one or more amino acidresidues other than the amino acid residue at the 365th position andhaving glucose dehydrogenase activity, wherein an amino acid residue ata position corresponding to the 365th position of the amino acidsequence is replaced with another amino acid residue, and the mutantglucose dehydrogenase shows an improved substrate specificity toglucose.(2) The mutant glucose dehydrogenase according to (1), which has theamino acid sequence of SEQ ID NO: 3 except for a position correspondingto the 365th position.(3) The mutant glucose dehydrogenase according to (1) or (2), whichshows a reduced reactivity to a disaccharide compared with a glucosedehydrogenase having a wild type amino acid residue at a positioncorresponding to the 365th position.(4) The mutant glucose dehydrogenase according to (3), wherein thedisaccharide is maltose.(5) The mutant glucose dehydrogenase according to (4), which shows areactivity to maltose in a degree of 20% or less of reactivity toglucose.(6) The mutant glucose dehydrogenase according to any one of (1) to (5),wherein the other amino acid residue is an amino acid residue selectedfrom phenylalanine, tyrosine, tryptophan and histidine residues.(7) The mutant glucose dehydrogenase according to any one of (1) to (6),wherein amino acid residue or residues corresponding to at least oneposition selected from the 324th, 326th, 333rd, 334th, 368th, 369th,376th, 377th, 418th, 419th, 436th, 433rd, 448th, 472nd, 475th, 525th and529th positions in the amino acid sequence of SEQ ID NO: 3 are replacedwith another or other amino acid residues.(8) The mutant glucose dehydrogenase according to (7), wherein theposition consists of at least one position selected from the 326th,472nd, 475th and 529th positions.(9) The mutant glucose dehydrogenase according to (8), wherein theposition is the 472nd position.(10) The mutant glucose dehydrogenase according to (8), wherein theposition is the 475th position.(11) The mutant glucose dehydrogenase according to (8), wherein theposition consists of both the 472nd position and the 475th position.(12) The mutant glucose dehydrogenase according to (8), wherein theposition is the 326th position.(13) The mutant glucose dehydrogenase according to (8), wherein theposition is the 529th position.(14) The mutant glucose dehydrogenase according to (9), wherein an aminoacid residue at a position corresponding to the 472nd position isreplaced with an amino acid residue selected from aspartic acid,glutamic acid, phenylalanine, tyrosine, isoleucine, asparagine andhistidine residues.(15) The mutant glucose dehydrogenase according to (10), wherein anamino acid residue at a position corresponding to the 475th position isreplaced with histidine or serine residue.(16) The mutant glucose dehydrogenase according to (12), wherein anamino acid residue corresponding to the 326th serine is replaced withglutamine or valine residue.(17) The mutant glucose dehydrogenase according to (13), wherein anamino acid residue corresponding to the 529th leucine is replaced withtyrosine, histidine or tryptophan residue.(18) A mutant glucose dehydrogenase having the amino acid sequence ofSEQ ID NO: 3 or an amino acid sequence of SEQ ID NO: 3 includingsubstitution, deletion, insertion or addition of one or more amino acidresidues and having glucose dehydrogenase activity, which includes

(i) substitution of other amino acid residue or residues for a residueor residues at a position or positions corresponding to at least oneposition selected from the 324th, 326th, 333rd, 334th, 365th, 368th,369th, 376th, 377th, 418th, 419th, 436th, 433rd, 448th, 525th and 529thpositions in the amino acid sequence of SEQ ID NO: 3,

(ii) substitution of aspartic acid residue for an amino acid residue ata position corresponding to the 472nd position, and

(iii) substitution of histidine residue for an amino acid residue at aposition corresponding to the 475th position, and shows improvedsubstrate specificity to glucose.

(19) The mutant glucose dehydrogenase according to (18), wherein theother amino acid residue or residues are selected from phenylalanine,tyrosine and tryptophan residues.

(20) The mutant glucose dehydrogenase according to (18), wherein theposition is the 326th position, and an amino acid residue at a positioncorresponding to the position is replaced with glutamine or valineresidue.

(21) The mutant glucose dehydrogenase according to (18), wherein theposition is the 529th position, and an amino acid residue at a positioncorresponding to the position is replaced with tyrosine, histidine ortryptophan residue.

(22) An FAD-binding type mutant glucose dehydrogenase containing theamino acid sequence of SEQ ID NO: 7.

(23) A mutant glucose dehydrogenase complex containing at least themutant glucose dehydrogenase according to any one of (1) to (22) and anelectron transfer subunit.

(24) The glucose dehydrogenase complex according to (23), wherein theelectron transfer subunit is cytochrome C.

(25) A DNA coding for the mutant glucose dehydrogenase according to anyone of (1) to (22).

(26) A microorganism harboring the DNA according to (25) and producingthe mutant glucose dehydrogenase according to any one of (1) to (22) orthe mutant glucose dehydrogenase complex according to (23).

(27) A glucose assay kit comprising the mutant glucose dehydrogenaseaccording to (1) to (22), the mutant glucose dehydrogenase complexaccording to (23), or the microorganism according to (26).

(28) A glucose sensor comprising the mutant glucose dehydrogenaseaccording to (1) to (22), the mutant glucose dehydrogenase complexaccording to (23), or the microorganism according to (26).

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows structure of a glucose sensor.

FIG. 2 shows reagent parts of a glucose sensor.

FIG. 3 shows a graph representing reactivity of a glucose sensor using amutant GDH for glucose.

FIG. 4 shows a graph representing reactivity of a glucose sensor using amutant GDH for maltose in the presence of glucose.

FIG. 5 shows a graph showing apparent blood sugar levels measured byusing glucose sensors using a wild type GDH or a mutant GDH.

BEST MODE FOR CARRYING OUT THE INVENTION

Hereafter, the present invention will be explained in detail.

The mutant GDH of the present invention is produced by introducing aspecific mutation into a wild type GDH.

Examples of the wild type GDH include GDHs produced by Burkholderiacepacia. Examples of the GDHs produced by Burkholderia cepacia includeGDHs produced by the Burkholderia cepacia KS1, JCM2800 and JCM2801strains. The KS1 strain was deposited at the independent administrativecorporation, National Institute of Advanced Industrial Science andTechnology, International Patent Organism Depositary (Tsukuba Central 6,1-1, Higashi 1-Chome, Tsukuba-shi, Ibaraki-ken, 305-8566, Japan) on Sep.25, 2000 and given an accession number FERM BP-7306. The JCM2800 andJCM2801 strains are stored at the independent administrativecorporation, RIKEN, Bioresource Center, Japan Collection ofMicroorganisms (JCM).

The nucleotide sequence of a chromosomal DNA fragment containing the GDHα-subunit gene and a part of the β-subunit gene of the KS1 strain isshown in SEQ ID NO: 1 (U.S. Patent Application No. 2004/0023330). Threeopen reading frames (ORF) exist in this nucleotide sequence, the secondand third ORFs from the 5′ end side code for the α-subunit (SEQ ID NO:3) and the 1-subunit (SEQ ID NO: 4), respectively. Further, it isinferred that the first ORF codes for the γ-subunit (SEQ ID NO: 2).Further, the nucleotide sequence of a fragment containing thefull-length β-subunit gene is shown in SEQ ID NO: 5. Further, the aminoacid sequence of the β-subunit is shown in SEQ ID NO: 6 (EP1498484A). Itis inferred that the amino acid numbers 1 to 22 in SEQ ID NO: 6correspond to a signal peptide. Although the first amino acid residuesare Val in SEQ ID NOS: 5 and 6, they are very likely to be Met and maybe eliminated after translation.

The mutant GDH of the present invention may consist of the α-subunitalone, a complex comprising the α-subunit and the β-subunit, or acomplex comprising the α-subunit, β-subunit and γ-subunit. The mutantGDH of the present invention is obtained by introducing a specificmutation into the α-subunit in any case, and may have a conservativemutation in addition to the above specific mutation. Further, the othersubunits may be of a wild type or have a conservative mutation. The term“conservative mutation” means a mutation that does not substantiallyaffect the GDH activity.

The mutant α-subunit of the present invention preferably has the aminoacid sequence of SEQ ID NO: 13 except that it includes the specificmutation described later. Further, the mutant α-subunit may have theaforementioned conservative mutation so long as it has the GDH activity.That is, it may be a protein having an amino acid sequence of SEQ ID NO:13 including substitution, deletion, insertion or addition of one ormore amino acid residues in addition to the aforementioned specificmutation. SEQ ID NO: 13 shows an amino acid sequence that can be encodedby the nucleotide sequence of SEQ ID NO: 11. However, the methionineresidue at the N-terminus may be eliminated after translation. Theaforementioned term “one or several” preferably means a number of 1 to10, more preferably 1 to 5, particularly preferably 1 to 3.

Further, the β-subunit typically has the amino acid sequence of SEQ IDNO: 16. However, so long as it functions as the β-subunit of GDH, it maybe a protein having an amino acid sequence of the amino acid numbers 23to 425 of SEQ ID NO: 16 including substitution, deletion, insertion oraddition of one or more amino acid residues. The aforementioned term“one or several” preferably means a number of 1 to 20, more preferably 1to 10, particularly preferably 1 to 5. The expression “functions as theGDH β-subunit” means to function as cytochrome C without degrading theenzymatic activity of GDH.

Specific examples of the wild type α-subunit gene include a DNAcontaining the nucleotide sequence corresponding to the nucleotidenumbers 764 to 2380 of SEQ ID NO: 11. Further, the α-subunit gene may bea DNA having the nucleotide sequence corresponding to the nucleotidenumbers 764 to 2380 in the nucleotide sequence of SEQ ID NO: 11 or a DNAwhich is hybridizable with a probe prepared from that sequence under astringent condition and codes for a protein having the GDH activity.

Further, specific examples of the β-subunit gene include a DNA havingthe nucleotide sequence corresponding to the nucleotide numbers 187 to1398 of SEQ ID NO: 9. Further, the β-subunit gene may be a DNA which hasthe nucleotide sequence corresponding to the nucleotide numbers 187 to1398 of SEQ ID NO: 9, or a DNA which is hybridizable with a probeprepared from that sequence under a stringent condition and codes for aprotein that can function as the β-subunit.

Examples of the aforementioned stringent condition include, for example,a condition under which DNAs having a homology of 70% or more,preferably 80% or more, more preferably 90% or more, particularlypreferably 95% or more, hybridize with each other, and it isspecifically exemplified by the condition of 1×SSC, 0.1% SDS at 60° C.

The α-subunit gene and the β-subunit gene can be obtained by, forexample, PCR using chromosomal DNA of the Burkhorderia cepacia KS1strain as a template. Primers for PCR can be prepared by chemicalsynthesis on the basis of the aforementioned nucleotide sequences.Further, they can also be obtained from chromosomal DNA of theBurkhorderia cepacia KS1 strain by hybridization using anoligonucleotide prepared on the basis of the aforementioned sequences asa probe. Further, variants thereof can also be similarly obtained fromother strains of Burkhorderia cepacia. Examples of the other bacterialstrains include the aforementioned JCM2800 and JCM2801 strains. Theα-subunits of GDHs produced by these strains have homologies of 95.4 and93.7%, respectively, to the α-subunit of the KS1 strain.

Further, GDHs produced by other microorganisms may also be used toprepare the mutant GDH of the present invention, so long as GDH having astructure and enzymatic characteristics similar to those of Burkhorderiacepacia GDH is chosen. Examples of such GDH include GDHs derived from(i) Burkholderia pseudomallei, (ii) Burkholderia mallei, and (iii)Ralstonia solanacearum ((i) and (ii): Proc. Natl. Acad. Sci. U.S.A. 101(39), 14240-14245 (2004), (iii): Nature 415 (6871), 497-502 (2002)).

The mutant GDH of the present invention shows improved substratespecificity to glucose, because it is obtained by adding the specificmutation to such a wild type GDH or GDH having a conservative mutationas described above. The “improved substrate specificity to glucose”include reduced reactivity to other monosaccharides, disaccharides andoligosaccharides such as maltose, galactose and xylose with thesubstantially same reactivity to glucose, and improved reactivity toglucose compared with reactivities to other saccharides. For example,even if the reactivity to glucose is reduced, if the reactivities toother saccharides are reduced in larger degrees, the substratespecificity for glucose is improved. Moreover, even if the reactivitiesto other saccharides are increased, if the substrate specificity toglucose is increased in a larger degree, the substrate specificity toglucose is improved. Specifically, for example, if improvement of thesubstrate specificity of the mutant enzyme relative to that of a wildtype enzyme (the substrate specificity is a ratio of specific activityfor saccharide other than glucose such as maltose to specific activityfor glucose, and the improvement is represented by the followingequation) is 10% or more, preferably 20% or more, more preferably 40% ormore, the substrate specificity shall be improved. For example, if thesubstrate specificity of a wild type enzyme is 60%, and the substratespecificity of a mutant GDH is 40%, the reactivity to other saccharidesother than glucose is reduced by 33%.Substrate specificity=(Specific activity for saccharides other thanglucose/Specific activity for glucose)×100Improvement of substrate specificity=(A−B)×100/AA: Substrate specificity of wild type enzymeB: Substrate specificity of mutant enzyme

Further, the reactivity (specific activity) for maltose of the mutantGDH is preferably 30% or less, more preferably 20% or less, of thereactivity (specific activity) for glucose.

Specific examples of the specific mutation include the followings.

(1) Substitution of another amino acid residue for the amino acidresidue at a position corresponding to the 365th position of the aminoacid sequence of SEQ ID NO: 3

(2) Substitution of another amino acid residue for the amino acidresidue at a position corresponding to the 365th position of the aminoacid sequence of SEQ ID NO: 3, and substitution of another or otheramino acid residues for at least one or arbitrary two or more amino acidresidues at position or positions corresponding to the 324th, 326th,333rd, 334th, 368th, 369th, 376th, 377th, 418th, 419th, 436th, 433rd,448th, 472nd, 475th, 525th and 529th positions in the amino acidsequence of SEQ ID NO: 3(3) (i) Substitution of another or other amino acid residues for aminoacid residue or residues at a position or positions corresponding to atleast one or arbitrary two or more positions selected from the 324th,326th, 333rd, 334th, 365th, 368th, 369th, 376th, 377th, 418th, 419th,436th, 433rd, 448th, 525th and 529th positions in the amino acidsequence of SEQ ID NO: 3, (ii) substitution of another amino acidresidue for an amino acid residue at a position corresponding to the472nd position, and (iii) substitution of another amino acid residue foran amino acid residue at a position corresponding to the 475th position.

Examples of the aforementioned specific mutation further include such amutation that the amino acid sequence of SEQ ID NO: 7 should becontained in the amino acid sequence of GDH. The amino acid sequence ofSEQ ID NO: 7 corresponds to the sequence of the 360th position to the366th position in GDH of SEQ ID NO: 3. It is considered that even if theGDH is an FAD-binding type GDH other than that of Burkhorderia cepacia,substrate specificity of a mutant GDH containing the amino acid sequenceof SEQ ID NO: 7 should be improved, if a corresponding wild type GDHdoes not contain the amino acid sequence of SEQ ID NO: 7.

Examples of the other amino acid residue mentioned in the above mutation(1) include those of amino acids other than serine, specifically,phenylalanine, tyrosine, aspartic acid, histidine, arginine, tryptophan,lysine, asparagine, leucine, cysteine, threonine, isoleucine, glycine,valine, methionine, glutamine, glutamic acid, alanine and proline. Amongthese, residues of phenylalanine, tyrosine, tryptophan and histidine arepreferred.

Examples of the other amino acid residue or residues mentioned in theabove mutation (2) include amino acid residues other than the amino acidresidues at those positions of a wild type GDH. Among the aforementionedpositions of the amino acid substitution, the 326th position, the 472ndposition, the 475th position, and the 529th position are preferred, andthe 472nd position and the 475th position are more preferred. Althoughthe amino acid residues at positions corresponding to the 472nd positionand the 475th position may be independently replaced, it is morepreferred that both are replaced.

The amino acid residue after the substitution at the 472nd position ispreferably aspartic acid, glutamic acid, phenylalanine, tyrosine,isoleucine, asparagine or histidine residue, particularly preferablyaspartic acid residue.

The amino acid residue after the substitution at the 475th position ispreferably histidine or serine residue, particularly preferablyhistidine residue.

The amino acid residue after the substitution at the 326th position ispreferably glutamine or valine residue.

The amino acid residue after the substitution at the 529th position ispreferably tyrosine, histidine or tryptophan residue.

Preferred embodiments of the mutation (3) mentioned above are similar tothose mentioned above.

The positions of the aforementioned amino acid substitution mutationsare those in SEQ ID NO: 3, that is, the amino acid sequence of the wildtype GDH α-subunit of the Burkholderia cepacia KS1 strain, and in a GDHα-subunit homologue or variant having an amino acid sequence includingsubstitution, deletion, insertion or addition of one or more amino acidresidues in the amino acid sequence of SEQ ID NO: 3 in addition to theaforementioned specific mutations, the positions are those correspondingto the positions of the aforementioned amino acid substitutionsdetermined by alignment with the amino acid sequence of SEQ ID NO: 3.For example, in a conservative GDH α-subunit variant having deletion ofone amino acid residue in the region of 1st to 364th positions, the365th position represents the 364th position in the variant.

Preferred embodiments of the mutation in the mutant GDH of the presentinvention are shown below (numerals represent positions in the aminoacid sequence, the amino acid residues represent amino acid residuesafter the substitution at the positions, and “+” means that two aminoacid substitutions are simultaneously included).

(A) 365Arg, 365Asn, 365Asp, 365Cys, 365Glu, 365Gly, 365His, 365Ile,365Leu, 365Met, 365Phe, 365Pro, 365Trp, 365Tyr, 365Val, 365Lys, 365Gln,365Thr, 365Ala

(B) 326Gln, 326Val, 326Arg

(C) 529His, 529Tyr, 529Trp

(D) 365Tyr+326Gln, 365Tyr+326Val, 365Tyr+326Arg, 365Tyr+472Phe,365Tyr+472Ile, 365Tyr+472Asn, 365Tyr+472Asp, 365Tyr+472H is,365Tyr+472Leu, 365Tyr+472Ser, 365Tyr+475Ser, 365Tyr+475His,365Phe+472Phe (E) 472Asp+475His+365Phe, 472Asp+475His+326Gln,472Asp+475His+326Thr, 472Asp+475His+326Val, 472Asp+475His+529Trp,472Asp+475His+529His, 472Asp+475His+529Tyr, 472Tyr+475His+365Phe,472Tyr+475His+365His, 472Tyr+475His+326Val, 472Ile+475His+326Gln(F) 472Asp+475His+529His+326Gln, 472Asp+475His+529Trp+326Gln

The inventors of the present invention compared the amino acid sequencesof the GMC oxidoreductase family enzymes using FAD as a coenzyme,sorbitol dehydrogenase of Gluconobacter oxydans (GenBank accessionAB039821), 2-ketoglutarate dehydrogenase of Erwinia herbicola (GenBankaccession AF068066), cellobiose dehydrogenase (CDH) of Phanerochaetechrysosporium (J. Mol. Biol., 315(3), 421-34 (2002)), cholesteroloxidase (COD) of Streptomyces species (J. Struct. Biol. 116(2), 317-9(1996)), and glucose oxidase of Penicillium amagasakiens (Eur. J.Biochem. 252, 90-99 (1998)), and found a region in which the FAD-bindingdomain and FAD-covering lid were conserved and a region in which prolinewas conserved, which is an amino acid residue involved in folding ofproteins. Then, they examined the possibility of improving substratespecificity by modifying sequences in the vicinity of the bordersbetween these regions and other regions. Specifically, R53 to H73, E88to A108, N308 to G336, K362 to A377, A391 to R497, and S509 to V539 wereexamined. As a result, they found some positions enabling improvement ofthe substrate specificity in the aforementioned regions.

A GDH α-subunit having a desired mutation can be obtained by introducinga nucleotide mutation corresponding to a desired amino acid mutationinto a DNA coding for the GDH α-subunit (α-subunit gene) bysite-directed mutagenesis and expressing the obtained mutant DNA byusing a suitable expression system. Further, a mutant GDH complex can beobtained by expressing a DNA coding for the mutant GDH α-subunittogether with a DNA coding for the β-subunit (β-subunit gene) or theβ-subunit gene and a DNA coding for the γ-subunit (γ-subunit gene).

For the introduction of a mutation into a DNA coding for the GDHα-subunit, a polycistronic DNA fragment coding for the GDH α-subunit,γ-subunit and β-subunit in this order may also be used.

Substrate specificities to sugars of the GDH α-subunit or the GDHcomplex introduced with the mutation can be determined by examiningreactivities to various sugars by the methods described in the examplesand comparing them with reactivities of a wild type GDH α-subunit or awild type GDH complex.

A polycistronic DNA fragment coding for the γ-subunit, α-subunit andβ-subunit in this order can be obtained by, for example, PCR usingchromosomal DNA of the Burkhorderia cepacia KS1 strain as a template andoligonucleotides having the nucleotide sequences of SEQ ID NOS: 8 and 9as primers (see the examples described later).

Examples of vectors used for obtaining the genes of GDH subunits,introduction of mutation, expression of the genes and so forth includevectors that function in Escherichia bacteria, and specific examplesthereof include pTrc99A, pBR322, pUC18, pUC118, pUC19, pUC119, pACYC184,pBBR122 and so forth. Examples of the promoters used for expression ofgenes include lac, trp, tac, trc, P_(L), tet, PhoA and so forth.Further, insertion of these genes into a vector and ligation of apromoter can be performed in one step by inserting the α-subunit gene orother subunit genes at a suitable site in an expression vectorcontaining the promoter. Examples of such an expression vector includepTrc99A, pBluescript, pKK223-3 and so forth.

Further, the α-subunit gene or other subunit genes may be incorporatedinto chromosomal DNA of a host microorganism in an expressible form.

Examples of the method for transforming a microorganism with arecombinant vector include, for example, the competent cell method usinga calcium treatment, protoplast method, electroporation and so forth.

Examples of the host microorganism include Bacillus bacteria such asBacillus subtilits, yeast such as Saccharomyces cerevisiae andfilamentous fungi such as Aspergillus niger. However, the hostmicroorganism is not limited to these examples, and host microorganismssuitable for producing foreign proteins can be used.

The mutant α-subunit, the mutant GDH complex, and the microorganismexpressing them of the present invention can be used as an enzymeelectrode of a glucose sensor or a component of a glucose assay kit. Aglucose sensor and glucose assay kit using the wild type GDH ofBurkhorderia cepacia are described in U.S. Patent No. 2004/0023330A1.The mutant GDH of the present invention can also be used in a similarmanner.

EXAMPLES

Hereafter, the present invention will be explained more specificallywith reference to examples. However, the present invention is notlimited to these examples.

Example 1 Plasmids Expressing GDH of Burkhorderia cepacia

As plasmids expressing GDH of Burkhorderia cepacia, a plasmid expressingthe GDH α-subunit and γ-subunit and a plasmid expressing the α-subunit,β-subunit and γ-subunit were prepared.

<1> Plasmid Expressing GDH α-Subunit and γ-Subunit

As a plasmid expressing the α-subunit and γ-subunit, plasmid pTrc99A/γ+αdescribed in WO02/036779 (corresponding to EP1331272A1, US2004023330A1CN1484703A) was used. This plasmid is a plasmid obtained by inserting aDNA fragment sequentially containing the GDH γ-subunit structural geneand the α-subunit structural gene isolated from chromosomal DNA of theBurkhorderia cepacia KS1 strain (FERM BP-7306) into the vector pTrc99A(Pharmacia) at the NcoI/HindIII site as a cloning site thereof. TheGDHγα gene in this plasmid is regulated by the trc promoter. pTrc99A/γ+αhas an ampicillin resistance gene.

<2> Plasmid Expressing GDH α-Subunit, β-Subunit and γ-Subunit

A plasmid expressing the GDH α-subunit, β-subunit and γ-subunit wasprepared as follows.

(1) Preparation of Chromosomal DNA from Burkhorderia cepacia KS1 Strain

A chromosomal gene was prepared from the Burkhorderia cepacia KS1 strainin a conventional manner. That is, the TL liquid medium (10 g ofpolypeptone, 1 g of yeast extract, 5 g of NaCl, 2 g of KH₂PO₄, 5 g ofglucose in 1 L, pH 7.2) was used, and cells of the strain was shakenovernight in the medium at 34° C. The grown cells were collected bycentrifugation. The cells were suspended in a solution containing 10 mMNaCl, 20 mM Tris-HCl (pH 8.0), 1 mM EDTA, 0.5% SDS and 100 μg/ml ofproteinase K and treated at 50° C. for 6 hours. To the mixture was addedan equal volume of phenol-chloroform, and the mixture was stirred atroom temperature for 10 minutes. Then, the supernatant was collected bycentrifugation. To the supernatant was added sodium acetate at a finalconcentration of 0.3 M, and 2-fold volume of ethanol was overlaid toprecipitate chromosomal DNA in the intermediate layer. The DNA wascollected with a glass rod, washed with 70% ethanol, and then dissolvedin a suitable volume of TE buffer to obtain a chromosomal DNA solution.

(2) Preparation of DNA Fragment Coding for GDH γ-Subunit, α-Subunit andβ-Subunit

A DNA fragment coding for the GDH γ-subunit, α-subunit and β-subunit wasamplified by PCR using the aforementioned chromosomal DNA as a templateand oligonucleotides having the following sequences as primers.

[Forward Primer]

5′-CATGCCATGGCACACAACGACAACAC-3′ (SEQ ID NO: 8)

[Reverse Primer]

5′-GTCGACGATCTTCTTCCAGCCGAACATCAC-3′ (SEQ ID NO: 9)

The C-terminus side of the amplified fragment was blunt-ended, theN-terminus side was digested with NcoI, and the fragment was ligated tosimilarly treated pTrc99A (Pharmacia). E. coli DH5a was transformed withthe obtained recombinant vector, and colonies grown on the LB agarmedium containing 50 μg/mL of ampicillin were collected. The obtainedtransformants were cultured in the liquid LB medium, plasmids wereextracted, and DNA fragments inserted in the plasmids were analyzed. Asa result, an inserted fragment of about 3.8 kb was confirmed. Thisplasmid was designated as pTrc99Aγαβ. The structural genes of GDH inthis plasmid are regulated by the trc promoter. pTrc99Aγαβ has anampicillin resistance gene and a kanamycin resistance gene.

Example 2 Search of Substrate Interaction Site of GDH α-Subunit byMutagenesis in GDH α-Subunit

(1) Mutagenesis at 472nd Position and 475th Position

The GDH α-subunit gene contained in pTrc99Aγαβ obtained in Example 1 wasmutagenized so that histidine residue and asparagine residue shouldsubstitute for the 472th alanine residue and the 475th aspartic acidresidue in the α-subunit encoded by the gene, respectively. This mutantis called 472D+475H mutant. It had already been confirmed by theinventors of the present invention that the substrate characteristicswere improved by such mutation.

Specifically, a commercially available site-directed mutagenesis kit(Stratagene, QuikChangeII Site-Directed Mutagenesis Kit) was used tosubstitute codon of aspartic acid (GAT) or glutamic acid (GAA) for thecodon of 475th asparagine (AAT) in the GDH α-subunit gene contained inthe plasmids pTrc99A/γ+α and pTrc99Aγαβ described in Example 1. Thefollowing oligonucleotides were used as primers.

[Primer for introducing A472D mutation] Forward primer: SEQ ID NO: 2245′-CGTGTTCAACGACGAATTCGATCCGAACAATCACATCACGG-3′ Reverse primer: SEQ IDNO: 225 5′-CCGTGATGTGATTGTTCGGATCGAATTCGTCGTTGAACACG-3′ [Primer forintroducing N475H mutation] Forward primer: SEQ ID NO: 2265′-AATTCGCGCCGAACCACCACATCACGGGCTC-3′ Reverse primer: SEQ ID NO: 2275′-GAGCCCGTGATGTGGTGGTTCGGCGCGAATT-3′(2) Mutagenesis at Position Other Than 472nd Position and 475th Position

The mutant gene coding for the 472D+475H mutant obtained above was usedto attain substitution of phenylalanine residue for amino acid residuesin the regions shown below. However, amino acid substitution was notperformed for the positions for which the wild type had phenylalanineresidue as the amino acid residue. The numerals used below indicatepositions in the amino acid sequence, and the alphabets before thenumerals indicate kinds of amino acids. For example, R53 representsarginine at the 53rd position.

(i) R53 to H73

(ii) E88 to A108

(iii) N₃₀₈ to G336

(iv) K362 to A377

(v) A391 to R497

(vi) S509 to V539

As the α-subunit gene as a target of the mutagenesis, the 472D+475Hmutant for which substrate characteristic improving effect was alreadyconfirmed was used aiming at obtaining synergistic effect by thecombination of mutations.

The sequences of the forward primers used for the aforementioned aminoacid residue substitution are shown below. Completely complementarystrands of the forward primers were used as the sequences of the reverseprimers.

In the notations of mutations, the numerals indicate positions in theamino acid sequence, the alphabets before the numerals indicate kinds ofamino acids before the amino acid substitution, and the alphabets afterthe numerals indicate amino acid residues after the amino acidsubstitution. For example, R53F represents substitution of phenylalaninefor arginine at the 53rd position.

PCR was performed by using the following reaction composition. After areaction at 95° C. for 30 seconds, a cycle of reactions at 95° C. for 30seconds, 55° C. for 1 minute and 68° C. for 8 minutes was repeated 15times. Then, after a reaction at 68° C. for 30 minutes, the reactionmixture was maintained at 4° C.

[Composition of reaction mixture] Template DNA (5 ng/μl)   2 μl (472D +475H introduced pTrc99A/γ + α and pTrc99Aγαβ) 10 × Reaction buffer   5μl Forward primer (100 ng/μl) 1.25 μl Reverse primer (100 ng/μl) 1.25 μldNTP   1 μl Distilled water 38.5 μl DNA polymerase   1 μl Total   50 μl

After PCR, 0.5 μl of DNA polymerase I was added to the reaction mixture,and the mixture was incubated at 37° C. for 1 hour to decompose thetemplate plasmid.

Competent cells of Escherichia coli DH5α (supE44, ΔlacU169(φ80lacZΔM15), hsdR17, recAi, endA1, gyrA96, thi-1, relA1) weretransformed with the obtained reaction mixture. Plasmids DNA wereprepared from several colonies grown on the LB agar medium (1% Bactotryptone, 0.5% yeast extract, 1% sodium chloride, 1.5% agar) containingampicillin (50 μg/ml) and kanamycin (30 μg/ml), and sequence analysiswas performed to confirm that the objective mutations had beenintroduced into the GDH α-subunit gene.

TABLE 1 Primers for substitution in R53-H73 region Amino acidsubstitution SEQ ID NO R53F 10 N54F 11 Q55F 12 P56F 13 D57F 14 K58F 15M59F 16 D60F 17 M62F 18 A63F 19 P64F 20 Y65F 21 P66F 22 S67F 23 S68F 24P69F 25 W70F 26 A71F 27 P72F 28 H73F 29

TABLE 2 Primers for substitution in E88-A108 region Amino acidsubstitution SEQ ID NO E88F 30 H89F 31 K90F 32 N92F 33 S93F 34 Q94F 35Y95F 36 I96F 37 R97F 38 A98F 39 V99F 40 G100F 41 G101F 42 T102F 43 T103F44 W104F 45 H105F 46 W106F 47 A107F 48 A108F 49

TABLE 3 Primers for substitution in N308-G336 region Amino acidsubstitution SEQ ID NO N308F 50 S309F 51 S310F 52 D311F 53 M312F 54V313F 55 G314F 56 R315F 57 N316F 58 L317F 59 M318F 60 D319F 61 H320F 62P321F 63 G322F 64 T323F 65 G324F 66 V325F 67 S326F 68 Y328F 69 A329F 70S330F 71 E331F 72 K332F 73 L333F 74 W334F 75 P335F 76 G336F 77

TABLE 4 Primers for substitution in K362-I377 region Amino acidsubstitution SEQ ID NO I362F 78 H363F 79 L364F 80 S365F 81 N366F 82L367F 83 S368F 84 R369F 85 I370F 86 D371F 87 Q372F 88 E373F 89 T374F 90Q375F 91 K376F 92 I377F 93

TABLE 5 Primers for substitution in A391-Y453 region Amino acidsubstitution SEQ ID NO A391F 94 Q392F 95 I393F 96 R394F 97 D395F 98R396F 99 S397F 100 A398F 101 R399F 102 Y400F 103 V401F 104 Q402F 105D404F 106 C405F 107 H407F 108 E408F 109 I409F 110 L410F 111 P413F 112E414F 113 N415F 114 R416F 115 I417F 116 V418F 117 P419F 118 S420F 119K421F 120 T422F 121 A423F 122 T424F 123 D425F 124 A426F 125 I427F 126G428F 127 I429F 128 P430F 129 R431F 130 P432F 131 E433F 132 I434F 133T435F 134 Y436F 135 A437F 136 I438F 137 D439F 138 D440F 139 Y441F 140V442F 141 K443F 142 R444F 143 G445F 144 A446F 145 A447F 146 H448F 147T449F 148 R450F 149 E451F 150 V452F 151 Y453F 152

TABLE 6 Primers for substitution in I477-R497 region Amino acidsubstitution SEQ ID NO I477F 153 T478F 154 G479F 155 S480F 156 T481F 157I482F 158 M483F 159 G484F 160 A485F 161 D486F 162 A487F 163 R488F 164D489F 165 S490F 166 V491F 167 V492F 168 D493F 169 K494F 170 D495F 171C496F 172 R497F 173

TABLE 7 Primers for substitution in S509-V539 region Amino acidsubstitution SEQ ID NO S509F 174 A510F 175 T511F 176 M512F 177 P513F 178T514F 179 V515F 180 G516F 181 T517F 182 V518F 183 N519F 184 V520F 185T521F 186 L522F 187 T523F 188 I524F 189 A525F 190 A526F 191 L527F 192A528F 193 L529F 194 R530F 195 M531F 196 S532F 197 D533F 198 T534F 299L535F 200 K536F 201 K537F 202 E538F 203 V539F 204

Example 3 Analysis of Substrate Specificity of Mutant GDHs

Mutant GDHs were produced by using the mutant GDH expressing plasmidsobtained in Example 2, and substrate specificities thereof wereexamined.

(1) Culture

Each of strains of Escherichia coli DH5α introduced with each mutationwas cultured overnight at 37° C. in 2 ml of the LB medium (containing 50μg/ml of ampicillin and 30 μg/ml of kanamycin) in an L-shaped tube withshaking. These culture broths were each inoculated into 150 ml of the LBmedium (containing 50 μg/ml of ampicillin and 30 μg/ml of kanamycin)contained in a 500-ml Sakaguchi flask, and the cells were cultured at37° C. with shaking. After 3 hours from the start of culture,isopropyl-β-D-thiogalactopyranoside (IPTG) was added at a finalconcentration of 0.1 mM, and the cells were further cultured for 2hours.

(2) Preparation of Crude Enzyme Samples

The cells were collected from each culture broth obtained as describedabove, washed, then suspended in 10 mM potassium phosphate buffer (PPB,pH 7.0) containing 1 ml of 0.2% Triton X-100 per 0.3 mg of wet cells,and disrupted by ultrasonication. This suspension was centrifuged (10000rpm, 10 min, 4° C.) to remove the residues, then the supernatant wasultracentrifuged (50,000 r.p.m., 60 min, 4° C.), and the obtainedsupernatant (water-soluble fraction) was used as a crude enzyme sample.Further, this sample was purified by usual hydrophobic chromatography(column: Octyl Sepharose, Amersham Biosciences) and ion exchangechromatography (Q-Sepharose, Amersham Biosciences) to obtain a purifiedenzyme sample. The objective enzyme fraction was determined by using GDHactivity as an index.

(3) Measurement of GDH Activity

To 8 μl of the aforementioned purified enzyme sample was added 8 μl of areagent for measuring activity (solution obtained by adding 10 mM PPBcontaining 0.2% (w/v) Triton X-100 to 12 μl of 600 mM methylphenazinemethosulfate (PMS) and 120 μl of 6 mM 2,6-dichrolophenol-indophenol(DCIP) to make a total volume of 480 μl). This mixture was preincubatedat each reaction temperature for one minute by using an aluminum blockthermostatic chamber, then 8 μl of a substrate (glucose or maltose) ateach concentration or distilled water was quickly added to the mixture,and the mixture was stirred. Absorbance at 600 nm as the DCIP-originatedabsorption wavelength was measured by using a spectrophotometer. Thefinal concentrations of the reagents, DCIP and PMS, were 0.06 and 0.6mM, respectively. The final concentration of the substrate was 5 mM.

The results are shown in Tables 8 to 14. The reaction ratio of the wildtype GDH was 48%.

TABLE 8 U/ml Reaction (culture medium) ratio Enzyme Glucose MaltoseMal/Glu A472D + N475H 2.02 0.28 13.8% R53F 3.34 0.42 12.6% N54F 2.190.32 14.4% Q55F 1.95 0.24 12.4% P56F 2.28 0.37 16.4% D57F 2.85 0.4214.9% K58F 1.57 0.20 12.8% M59F 1.94 0.29 14.8% D60F 2.62 0.36 13.9%M62F 0.85 0.16 19.2% A63F 0.66 0.12 18.6% P64F 1.79 0.30 16.9% Y65F 0.290.16 54.0% P66F 0.12 0.12 100.0% S67F 0.70 0.20 28.0% S68F 2.23 0.2611.6% P69F 0.60 0.09 15.1% W70F 2.64 0.32 12.2% A71F 2.90 0.31 10.7%P72F 0.05 0.06 112.7% H73F 3.25 0.39 12.1%

TABLE 9 U/ml Reaction (culture medium) ratio Enzyme Glucose MaltoseMal/Glu E88F 1.14 0.16 16.6% H89F 1.83 0.28 17.1% K90F 1.19 0.14 14.6%N92F 1.11 0.17 18.4% S93F 2.47 0.38 16.7% Q94F 1.16 0.14 15.0% Y95F 1.890.20 12.5% I96F 0.53 0.06 17.7% R97F 0.73 0.10 17.6% A98F 2.47 0.2411.0% V99F 0.40 0.04 17.4% G100F 0.07 0.02 50.5% G101F −0.02 −0.01108.3% T102F −0.02 −0.02 126.7% T103F −0.03 −0.01 203.0% W104F 0.52 0.0414.4% H105F 0.01 0.01 112.7% W106F 0.29 0.03 20.4% A107F 0.52 0.03 12.4%A108F 0.00 0.00 104.1%

TABLE 10 U/ml Reaction (culture medium) ratio Enzyme Glucose MaltoseMal/Glu N308F 0.03 0.04 104.7% S309F 0.03 0.03 94.4% S310F 0.04 0.06144.8% D311F 0.76 0.08 10.3% M312F 0.19 0.07 35.4% V313F 0.04 0.04103.8% G314F 0.15 0.08 50.6% R315F 0.01 0.01 89.5% N316F 0.16 0.07 44.4%L317F 0.10 0.07 70.4% M318F 0.04 0.01 26.6% D319F 0.03 0.02 53.8% H320F1.30 0.23 17.9% P321F 2.43 0.40 16.6% G322F 0.04 0.04 98.9% T323F 0.620.10 16.5% G324F 3.34 0.20 6.0% V325F 0.08 0.09 114.1% S326F 2.45 0.176.7% Y328F 0.03 0.03 97.2% A329F 0.69 0.09 12.4% S330F 0.39 0.07 17.3%E331F 1.86 0.17 9.4% K332F 0.81 0.10 12.8% L333F 3.05 0.26 8.4% W334F4.18 0.29 7.0% P335F 0.10 0.02 22.4% G336F 0.02 0.00 0.5%

TABLE 11 U/ml Reaction (culture medium) ratio Enzyme Glucose MaltoseMal/Glu I362F 0.11 0.10 88.2% H363F 0.47 0.10 20.4% L364F 3.30 0.3310.1% S365F 2.89 0.04 1.2% N366F 0.04 0.03 75.8% L367F 4.30 0.44 10.2%S368F 3.88 0.25 6.4% R369F 3.58 0.25 7.1% I370F 1.30 0.12 8.9% D371F2.73 0.35 12.9% Q372F 3.98 0.36 8.9% E373F 1.60 0.21 12.9% T374F 1.910.18 9.4% Q375F 3.59 0.39 10.8% K376F 2.83 0.20 6.9% I377F 3.05 0.237.6%

TABLE 12 U/ml Reaction (culture medium) ratio Enzyme Glucose MaltoseMal/Glu A391F 2.07 0.22 10.9% Q392F 2.21 0.22 10.2% I393F 1.76 0.1911.1% R394F 0.73 0.13 17.4% D395F 1.39 0.17 12.0% R396F 1.20 0.14 11.9%S397F 0.75 0.08 10.8% A398F 2.11 0.22 10.3% R399F 1.98 0.20 10.1% Y400F2.35 0.24 10.0% V401F 0.57 0.11 19.2% Q402F 0.58 0.07 12.3% D404F 0.280.08 29.2% C405F 0.06 0.06 104.9% H407F 0.08 0.06 67.3% E408F 0.08 0.0897.0% I409F 0.16 0.04 25.0% L410F 0.08 0.07 85.7% P413F 0.05 0.04 72.9%E414F 1.22 0.15 12.6% N415F 0.03 0.04 119.5% R416F 0.18 0.03 17.1% I417F0.05 0.05 86.6% V418F 2.66 0.19 7.3% P419F 1.74 0.10 5.7% S420F 0.450.04 8.5% K421F 0.42 0.06 15.0% T422F 1.98 0.18 9.1% T424F 2.27 0.2611.6% D425F 0.02 0.02 124.5% A426F 1.75 0.19 10.8% I427F 1.95 0.25 12.8%G428F 0.25 0.14 56.1% I429F 0.46 0.06 12.0% P430F 0.41 0.05 11.2% R431F1.80 0.16 8.7% P432F 0.19 0.11 56.4% E433F 1.31 0.14 10.4% I434F 0.130.05 41.2% T435F 1.16 0.13 11.0% Y436F 4.35 0.26 6.1% A437F 2.44 0.229.0% I438F 0.07 0.03 39.8% D439F 0.01 0.01 73.0% D440F 0.03 0.04 155.6%Y441F 0.03 0.04 119.5% V442F 0.01 0.01 175.0% K443F 4.64 0.27 5.9% R444F2.41 0.26 10.8% H448F 0.87 0.07 8.3% T449F 2.47 0.30 12.0% R450F 0.040.05 112.7% E451F 2.15 0.22 10.0% V452F 1.17 0.18 15.3% Y453F 0.17 0.18106.1%

TABLE 13 U/ml Reaction (culture medium) ratio Enzyme Glucose MaltoseMal/Glu I477F 0.83 0.20 24% T478F 0.03 0.03 104% G479F 0.05 0.05 96%S480F 0.08 0.11 144% T481F 0.01 0.00 −20% I482F 0.01 −0.01 −200% M483F2.54 0.22 9% G484F 0.01 0.01 116% A485F 0.05 0.06 116% D486F 0.77 0.1014% A487F 0.10 0.07 67% R488F 0.08 0.07 88% D489F 1.57 0.18 11% S490F0.03 0.04 149% V491F 0.05 0.04 81% V492F 0.08 0.08 109% D493F 0.05 0.0377% K494F 1.32 0.26 20% D495F 0.10 0.07 74% C496F 0.04 0.07 150% R497F0.19 0.13 68%

TABLE 14 U/ml Reaction (culture medium) ratio Enzyme Glucose MaltoseMal/Glu S509F 0.16 0.04 24% A510F 0.20 0.05 25% T511F 0.18 0.04 20%M512F 2.18 0.25 11% P513F 3.50 0.35 10% T514F 0.01 0.00 41% V515F 0.040.03 78% G516F 0.02 0.01 76% T517F 0.03 0.02 83% V518F 0.02 0.01 73%N519F 0.04 0.03 78% V520F 0.02 0.01 58% T521F −0.01 −0.01 168% L522F0.01 0.00 −29% T523F 0.01 0.00 24% I524F 0.01 −0.01 −110% A525F 3.200.24 7% A526F 0.02 0.00 23% L527F 0.29 0.10 34% A528F 0.00 −0.01 350%L529F 3.32 0.30 9% R530F 0.40 0.10 26% M531F 0.10 0.04 42% S532F 0.000.00 23% D533F 0.03 0.01 27% L537F 0.45 0.10 22% E538F 3.08 0.52 17%V539F 1.52 0.29 19%

As a result, it was found that 472D+475H+365F was an extremely effectivemutation, which reduced reactivity to maltose to a level of 1%, whilemaintaining the reactivity to glucose.

Among mutations other than the mutation at the 365th position, mutationsfor substituting phenylalanine for G324, S326, L333, W334, S368, R369,K376, I377, V418, P419, Y436, K433, H448, A525 and L529 reduced thereactivity to maltose to 60% or less compared with that before themutagenesis, and thus they were effective.

Example 4 Examination of Mutagenesis at 365th Position

From the results obtained in Example 3, it was estimated that the 365thposition was a very effective position for reducing the reactivity tomaltose. Therefore, it was decided to examine this position in detail.

Specifically, the substrate characteristic improving effect of singlemutagenesis at the 365th position of the α-subunit gene was examined.

Mutagenesis was carried out at the 365th position of the wild type GDHα-subunit gene contained in pTrc99Aγαβ, and the substrate specificity ofthe mutated enzyme was evaluated. The mutagenesis was carried out in thesame manner as that used in Example 2.

Forward primers for the mutagenesis were as follows. Completelycomplementary strands of the forward primers were used as reverseprimers.

TABLE 15 Primers for substitution at S365 position Amino acidsubstitution SEQ ID NO S365G 205 S365A 206 S365V 207 S365L 208 S365I 209S365M 210 S365P 211 S365F 212 S365W 213 S365T 214 S365N 215 S365Q 216S365Y 217 S365C 218 S365K 219 S365R 220 S365H 221 S365D 222 S365E 223

Example 5 Analysis of Substrate Specificity of Mutant GDHs

By using the mutant GDH expressing plasmids obtained in Example 4,mutant GDHs were prepared, and substrate specificities thereof wereexamined in the same manner as in Example 3. The enzymatic activity wasexamined by using crude enzyme samples. The specific activity forglucose, specific activity for maltose and reaction ratio (specificactivity for maltose/specific activity for glucose, unit is U/ml.) ofeach mutant GDH are shown in Table 16. As for the substrateconcentration, the evaluation was performed at 5 mM and 10 mM.

TABLE 16 Single mutagenesis U/ml (culture medium) 5 mM 10 mM GlucoseMaltose Mal/Glc Glucose Maltose Mal/Glc Wild 2.87 0.77 26.9% 2.52 1.2248.2% type S365F 3.02 0.46 15.4% 4.59 1.00 21.7% S365D 2.77 0.41 14.8%4.73 0.87 18.4% S365H 0.90 0.04 4.3% 1.49 0.12 8.1% S365R 1.09 0.08 6.9%1.96 0.28 14.1% S365W 0.47 0.01 2.3% 1.17 0.02 2.0% S365Y 1.42 0.02 1.6%2.46 0.05 2.0% S365G 2.51 0.42 16.9% 3.80 0.81 21.3% S365E 0.55 0.047.6% 0.98 0.19 19.2% S365I 1.00 0.08 7.6% 1.56 0.16 10.5% S365P 0.620.06 9.7% 1.29 0.17 13.3% S365L 0.97 0.07 7.4% 1.59 0.19 11.7% S365N1.90 0.23 12.3% 2.92 0.43 14.8% S365K 1.61 0.13 7.9% 2.35 0.26 10.9%S365Q 1.68 0.19 11.4% 3.53 0.44 12.6% S365M 2.10 0.29 13.7% 3.99 0.5614.1% S365T 1.76 0.23 13.1% 3.19 0.56 17.4% S365A 3.14 0.49 15.7% 4.800.29 6.0% S365C 1.79 0.19 10.4% 2.74 0.20 7.4% S365V 2.20 0.26 12.0%3.03 0.62 20.4%

As a result, as for the 365th position, the substrate characteristicimproving effect was observed for substitutions of all the amino acidresidues of 19 types other than the substitution of serine residue inthe wild type. Specifically, all the amino acid substitutions reducedthe reactivity to maltose by 50% or more compared with the wild type. Inparticular, after the substitutions of tyrosine and tryptophan residues,the reactivity to maltose was 2% or less at both the substrateconcentrations, 5 mM and 10 mM, whereas the reaction ratio of the wildtype was around 40%. That is, the substitutions reduced the reactivityto maltose by 90% or more, and thus extremely significant substratecharacteristic improving effects were observed.

Example 6 Examination of Combination Effect of 365th Position and OtherPositions

As shown by the results of Examples 2 and 5, the effect was confirmed atleast for the combinations of the mutation at the 365th position withthe mutations 365F and 472D+475H, and single mutagenesis by all theamino acid substitutions only at the 365th position. Therefore, itbecame clear that the 365th position was an extremely highly effectiveposition for improving the substrate characteristics. Therefore, aimingat further improvement of the substrate characteristics by combining theamino acid substitution at the 365th position and amino acidsubstitutions at other positions, further studies were conducted.Specifically, double mutagenesis at the 365th position and any of the326th, 529th and 472nd positions, and triple mutagenesis at the 365th,472nd, and 475th positions were examined.

TABLE 17 Double mutagenesis U/ml (culture medium) 5 mM 10 mM GlucoseMaltose Mal/Glc Glucose Maltose Mal/Glc S365Y 1.42 0.02 1.6% 2.46 0.052.0% (single mutation) S365F 3.02 0.46 15.4% 4.59 1.00 21.7% (singlemutation) 365Y + 326C 2.56 0.02 1.0% 3.71 0.06 1.5% 365Y + 326Q 2.280.01 0.6% 3.32 0.03 1.0% 365Y + 326T 1.76 0.02 1.2% 2.64 0.06 2.3%365Y + 326V 1.55 0.01 0.5% 2.47 0.01 0.5% 365Y + 326G 2.36 0.03 1.4%3.32 0.09 2.6% 365Y + 326E 1.90 0.02 1.1% 2.82 0.05 1.7% 365Y + 326K2.98 0.06 2.0% 4.22 0.17 4.0% 365Y + 326Y 1.60 0.02 1.3% 2.33 0.04 1.8%365Y + 326H 1.27 0.02 1.5% 1.84 0.04 2.2% 365Y + 326R 1.84 0.01 0.8%2.63 0.03 1.0% 365Y + 472F 1.35 0.01 0.8% 2.04 0.02 1.1% 365F + 326T0.54 0.10 18.4% 6.84 1.38 20.1% 365F + 326G 0.46 0.08 17.8% 5.51 1.3724.9% 365F + 326E 2.44 0.33 13.6% 3.06 0.61 19.8% 365F + 326R 3.14 0.4915.5% 3.83 0.81 21.1% 365F + 472F 1.51 0.01 0.6% 2.28 0.02 1.0% 365Y +475H 2.07 0.027 1.0% 3.62 0.044 1.5% 365Y + 475S 3.41 0.066 1.9% 4.540.116 2.6% 365Y + 472G 2.60 0.064 2.5% 4.76 0.078 1.6% 365Y + 472I 1.310.008 0.6% 2.04 0.020 1.0% 365Y + 472N 2.19 0.018 0.8% 3.42 0.052 1.5%365Y + 472D 1.33 0.009 0.6% 2.08 0.004 0.2% 365Y + 472E 2.99 0.068 2.3%5.27 0.089 1.7% 365Y + 472R 1.77 0.030 1.7% 2.87 0.025 0.9% 365Y + 472V3.19 0.073 2.3% 5.10 0.095 1.9% 365Y + 472C 0.74 0.023 3.1% 1.07 0.0181.7% 365Y + 472H 1.04 0.002 0.2% 1.63 0.005 0.3% 365Y + 472L 3.21 0.0531.6% 4.85 0.062 1.3% 365Y + 472M 1.17 0.019 1.6% 1.87 0.029 1.5% 365Y +472F 1.67 0.029 1.7% 2.99 0.043 1.4% 365Y + 472P 0.44 0.080 18.1% 0.720.048 6.7% 365Y + 472S 3.70 0.079 2.1% 4.74 0.087 1.8% 365Y + 472W 0.840.046 5.5% 1.63 0.044 2.7% 365Y + 472Y 0.98 0.051 5.2% 1.85 0.042 2.3%

TABLE 18 Triple mutagenesis U/ml (culture broth) 5 mM 10 mM GlucoseMaltose Mal/Glc Glucose Maltose Mal/Glc 472D + 475H 1.54 0.19  13% 2.330.34  14% +S365F 1.33 0.01 0.7% 2.44 0.02 0.9% +S365D 0.25 0.01 2.1%0.64 0.01 1.9% +S365H 0.48 0.01 1.9% 0.85 0.02 2.5% +S365R 0.15 0.019.6% 0.54 0.04 7.5% +S365W 0.30 0.01 3.0% 0.54 0.03 4.8% +S365Y 0.970.03 2.9% 1.66 0.08 5.1% +S365V 0.07 0.01 8.3% 0.16 0.01 6.8% 472Y +475H 1.30 0.06 4.5% 2.06 0.15 7.3% +S365F 1.15 0.01 0.7% 1.83 0.01 0.6%+S365D 0.27 0.01 4.9% 0.55 0.05 9.5% +S365H 0.49 0.01 1.1% 0.95 0.011.5% +S365R 0.33 0.02 5.0% 0.83 0.06 6.9% +S365W 0.22 0.01 2.3% 0.370.01 3.0% +S365Y 1.09 0.04 4.0% 1.88 0.13 6.8% +S365V 0.79 0.02 2.0%472F + 475S 0.67 0.05 7.6% 1.35 0.11 8.0% +S365F 0.88 0.06 6.7% 1.190.08 7.1% +S365D 0.35 0.01 4.2% 0.97 0.03 3.5% +S365H 0.04 0.00 9.7%0.13 0.00 3.4% +S365R 0.39 0.02 4.9% 0.92 0.04 4.5% +S365W 0.10 0.002.9% 0.24 0.00 1.3% +S365Y 0.36 0.01 4.1% 0.56 0.02 3.0% 472F + 475H2.75 0.35 12.8%  3.90 0.63 16.1%  +S365Y

It was found that the substrate characteristics were synergisticallyimproved by the combinations with all the positions examined, and itbecame clear that the substrate characteristics could be furtherimproved by a combination of a mutation at the 365th position and aminoacid substitution at another position improving the substratecharacteristics.

Example 7 Examination of Combination Effect of 472nd and 475th Positionswith Other Positions

From the results of Example 6, it was suggested that a combination ofamino acid substitutions showing a substrate characteristic improvingeffect might synergistically improve the substrate characteristics.

Therefore, combination effect of a mutation at the 472 or 475thposition, for which the substrate characteristic improving effect wasalready confirmed by the inventors of the present invention, and amutation at a position other than the 365th position was examined.

Specifically, combinations of mutations of 472D+475H, which showed thehighest substrate characteristic improving effect among the combinationsof mutations at the 472nd and 475th position, and a mutation at the326th position or the 529th position were examined. Moreover, othermutations at the 472nd and 475th positions were also examined.

TABLE 19 Triple mutagenesis U/ml (culture broth) 5 mM 10 mM GlucoseMaltose Mal/Glc Glucose Maltose Mal/Glc 472D + 475H 1.54 0.19  13% 2.330.34  14% +326C 1.27 0.03 2.4% 2.08 0.11 5.2% +326Q 1.22 0.02 1.8% 2.040.09 4.2% +326T 1.29 0.02 1.8% 2.13 0.10 4.7% +326V 0.69 0.01 2.0% 1.100.03 3.0% +326G 1.98 0.07 3.6% 2.99 0.22 7.3% +326L 0.09 0.00 3.1% 0.240.01 3.4% +326E 0.34 0.00 0.8% 0.69 0.02 2.9% +326I 0.35 0.01 1.7% 0.770.06 7.5% +326K 1.55 0.05 3.1% 1.87 0.14 7.2% 472D + 475H + 326Q 1.200.02 1.7% 1.87 0.07 3.9% 472E + 475H + 326Q 1.55 0.08 5.5% 2.69 0.269.7% 472H + 475H + 326Q 0.10 0.02 23.6%  0.17 0.06 32.0%  472I + 475H +326Q 1.05 0.03 2.6% 1.81 0.08 4.5% 472L + 475H + 326Q 2.17 0.15 6.7%3.41 0.32 9.3% 472M + 475H + 326Q 3.07 0.28 9.0% 4.99 0.53 10.7%  472N +475H + 326Q 2.45 0.24 9.7% 4.01 0.57 14.3%  472W + 475H + 326Q 2.82 0.4415.8%  4.37 0.89 20.4%  472Y + 475H + 326Q 1.73 0.07 4.3% 3.03 0.19 6.1%472D + 475H + 326V 0.69 0.01 2.0% 1.10 0.03 3.0% 472E + 475H + 326V 1.900.12 6.2% 2.88 0.25 8.8% 472H + 475H + 326V 1.07 0.08 7.4% 1.91 0.199.7% 472I + 475H + 326V 2.90 0.26 9.0% 4.35 0.69 15.9%  472L + 475H +326V 1.32 0.05 4.1% 2.00 0.12 6.1% 472M + 475H + 326V 3.52 0.32 9.0%5.64 0.41 7.4% 472N + 475H + 326V 2.85 0.25 8.7% 4.48 0.49 10.9%  472W +475H + 326V 2.71 0.38 14.0%  4.56 0.74 16.2%  472Y + 475H + 326V 0.990.01 1.2% 1.79 0.05 2.5%

TABLE 20 Triple mutagenesis U/ml(culture broth) 5 mM 10 mM GlucoseMaltose Mal/Glc Glucose Maltose Mal/Glc 472D + 475H 1.54 0.19 12.5% 2.330.34 14.5% +529F 2.13 0.29 13.7% 3.11 0.41 13.3% +529Y 0.21 0.02 8.3%0.42 0.03 7.9% +529H 1.36 0.11 7.8% 2.24 0.21 9.4% +529W 0.49 0.03 5.1%0.78 0.04 5.7%

TABLE 21 Quadruple mutagenesis U/ml (culture broth) 5 mM 10 mM Mal/ Mal/Glucose Maltose Glc Glucose Maltose Glc 472D + 475H + 0.49 0.01 1.9%0.93 0.03 2.8% 529H + 326Q 472D + 475H + 0.45 0.02 4.7% 0.72 0.07 10.1%529W + 326Q

The synergistic effect was also confirmed for combinations of mutationsat the 472 and 475th positions and other positions. In particular, thequadruple mutagenesis at the 472nd, 475th, 326th and 529th positionsshowed a high reducing effect for reactivity to maltose, and it was alsodemonstrated by this result that the substrate characteristics could besynergistically improved by combinations of the amino acid substitutionshaving a substrate characteristic improving effect.

Example 7 Preparation of Purified Enzymes

Some mutant GDHs among those for which improvement of the substratespecificity was observed in Examples 5 and 6 were purified. The methodwas the same as that described in Example 3. Specific activities (U/mg)for glucose of the purified enzymes are shown in Table 8.

As a result, it became clear that the mutations including S365Ymaintained about 90% of the specific activity for glucose, and thus theywere preferred mutations from the viewpoint of glucose measurement.Moreover, it also became clear that the S326Q mutation had an effect ofincreasing the specific activity for glucose.

TABLE 22 Specific Enzyme activity Wild type 1490 U/mg-p 472Y + 475H +326V 1547 U/mg-p 365Y 1234 U/mg-p 326Q + 365Y 1316 U/mg-p 472D + 475H +365F  477 U/mg-p 472D + 475H + 529W  963 U/mg-p 472D + 475H + 529H  961U/mg-p 472D + 475H + 326Q 1316 U/mg-p 472D + 475H + 326V 1157 U/mg-p

Example 8 Preparation of Calorimetric Sensor for Measuring Blood SugarLevels Using Mutant GDHs

Colorimetric sensors for measuring blood sugar level were prepared byusing the mutant GDHs obtained in Example 7.

A glucose sensor having a basic structure shown in FIG. 1 was prepared.That is, the aforementioned glucose sensor had a configuration that atransparent cover 4A (material: PET) was laminated on a transparent baseplate 2A via a spacer 3, and the capillary 5A was defined by theelements 2A to 4A. The dimension of the capillary 5A was 1.3 mm×9 mm×50μm (FIG. 1). The transparent base plate 2A and the transparent cover 4Awere formed with PET having a thickness of 250 μm, and the spacer 3A wasformed with a black double-sided tape.

The glucose sensor had a first reagent part, a second reagent part and athird reagent part shown in FIG. 2, and ingredients and coating amountsfor each part are shown in Table 23. In the table, “Ru” represents aruthenium hexaammine complex (Ru(NH₃)₆Cl₃), CHAPS represents3-[(3-cholamidopropyl)dimethylammonio]propanesulfonic acid, ACESrepresents N-(2-acetamido)-2-aminoethanesulfonic acid, and MTTrepresents 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazoliumbromide.

TABLE 23 First reagent part Material solution for reagent partcontaining electron transfer substance (solvent is water) Ru Coatingamount 200 mM 0.2 μl Second reagent part Material solution for reagentpart containing enzyme (solvent is water) Enzyme Sucrose Coatingconcentration CHAPS monolaurate ACES (pH 7.5) amount 15 KU/ml 0.20%0.05% 75 mM 0.1 μl Third reagent part Material solution for reagent partcontaining color developer (solvent is water) MTT Acrylamide MethanolCoating amount 60 mM 0.40% 50% 0.2 μl

An assay sample was supplied to the capillary of the aforementionedglucose sensor, and thereafter absorbance was repeatedly measured every0.1 second to prepare a time course of absorbance. For each measurementof absorbance, the third reagent part was irradiated with light alongthe direction of the height of the capillary, and upon the irradiation,light that transmitted through the glucose sensor was received. Thelight irradiation was attained by irradiation with light of 630 nm usinga light-emitting diode. The transmitted light was received with aphotodiode.

As the assay sample, blood added with glucose was used. Blood samples ofwhich hematocrit was adjusted to 42% were added with glucose atconcentrations of 0, 100, 200, 400, 600 and 800 mg/dl and used toevaluate linearity of the glucose sensor. The linearity was evaluated byplotting absorbance values at endpoints 5 seconds after the introductionof the assay samples into the sensor. The results are shown in FIG. 3.

Further, blood samples of which hematocrit was adjusted to 45% andglucose concentration was adjusted to 45 mg/dl were further added withmaltose at concentrations of 0, 100, 200 and 300 mg/dl, and used toevaluate influence of maltose. The results are shown in FIG. 4. Thelinearity was evaluated by plotting absorbance values at endpoints 5seconds after the introduction of the assay samples also in this test.

When the linearity was evaluated by using glucose as the substrate, theabsorbance increased in a concentration-dependent manner with all theenzymes, and it can be seen that glucose can be measured. In particular,the mutant GDHs including the mutation 365Y or 326Q365Y, which is amutation including a mutation at the 365th position, showed improvedlinearity compared with that shown by the wild type, and thus it issuggested that they enables the measurement up to a higher concentration(around 600 mg/dl).

As for the influence of maltose, when maltose was added to the samplescontaining 45 mg/dl of glucose, absorbance increased in a maltoseconcentration-dependent manner with the wild type, which suggestedstrong reaction with maltose. On the other hand, with the sensors usingthe mutant enzymes, the maltose concentration-dependent increase of theabsorbance was suppressed, showing less influence of maltose. Theresults obtained by converting these data into apparent blood sugarelevation values are shown in FIG. 5 and Table 24. In the sensor usingthe wild type enzyme, a hypoglycemic level (45 mg/dl of glucose) wasapparently indicated as a normal value (138 mg/dl of glucose) due tocontamination of maltose. On the other hand, in the sensors using themodified GDHs, the apparent blood sugar level elevated to only 60 mg/dlat most, even when the sample is contaminated with up to 300 mg/dl ofmaltose, and thus it can be said that the influence was significantlysuppressed. In conclusion, it is suggested that mutations including amutation at the 365th position are the most suitable in view ofreactivity to glucose (linearity) and influence of maltose.

TABLE 24 Maltose addition Wild type 365Y 326Q + 365Y 472D + 475H + 365F 0 45 45 45 45 100 75 45 47 46 200 113 48 47 48 300 138 50 58 54 Maltose472Y + 472D + 472D + 472D + 472D + addition 475H + 326V 475H + 529W475H + 529H 475H + 326Q 475H + 326V  0 45 45 45 45 45 100 53 45 46 45 45200 60 46 47 49 47 300 60 47 49 50 53 *Unit: mg/dl

As clearly seen from the above results, in the glucose sensors using themutant GDHs, reactivity to maltose was significantly decreased eventhough the linearity was maintained to an extent comparable to or hitherthan that of the wild type. It can be said that if these glucose sensorsusing the mutant GDHs are used, erroneous values are not observed at theupper limit of blood maltose level administered at hospitals or thelike, 200 mg/dl, and even at 300 mg/dl higher than the upper limit, ahypoglycemic value (50 mg/dl or less) is not judged as a normal value orhyperglycemic level, and thus safe therapeutic treatment can beconducted. Further, since GDHs do not react with dissolved oxygen asdescribed above, accurate diagnosis and treatment of diabetic patientscan be conducted by providing sensors using these mutant GDHs.

Example 9 Evaluation of Purified Enzymes Based on SV Plot

SV plots were obtained for the 365Y and 326Q+365Y mutated GDHs, forwhich substrate specificity improving effect was observed in Examples 5and 6, as purified enzymes.

As a result, it was confirmed that the reaction ratios (specificactivity for maltose/specific activity for glucose) of the purifiedenzymes were also became markedly lower than that of the wild type andthus improved at all the examined substrate concentrations. Further,since the results were substantially consistent with the measurementresults obtained by using the crude enzyme solutions, sufficientfeasibility for evaluation of modified enzymes using crude enzymes couldbe confirmed. In addition, since the blood maltose level elevates up to200 mg/dl even at most, attentions were paid particularly to thereaction ratios at the substrate concentrations of 10 mM (360 mg/dl) and5 mM (180 mg/dl). As a result, the maltose/glucose reaction ratio of theS365Y mutated GDH was 0.1% in this concentration range, and thus it wassuggested that it hardly reacted with maltose.

Moreover, it also became clear that use of the mutation 326Q in additionto the mutation 365Y can increase the specific activity for glucose, andrelatively decrease the reactivity to maltose.

TABLE 25 Evaluation of enzymatic characteristics 40 20 10 5 2.5 1.250.625 mM specific activity for glucose wild type 1543.7 1490.0 1388.11223.4 925.1 604.6 346.2 U/mg-p 365Y 1464.4 1234.0 970.8 661.1 397.2218.2 101.8 U/mg-p 326Q365Y 1525.8 1316.0 1022.4 686.4 436.8 238.8 114.7U/mg-p specific activity for maltose wild type 658.1 501.9 335.3 194.385.9 27.9 5.4 U/mg-p 365Y 31.1 8.1 1.4 0.5 0.2 0.1 0.0 U/mg-p 326Q365Y31.2 7.2 1.4 0.3 0.4 0.0 0.0 U/mg-p malsose/glucose (reaction ratio)wild type 42.6% 33.7% 24.2% 15.9% 9.3% 4.6% 1.6% % 365Y 2.1% 0.7% 0.1%0.1% 0.0% 0.1% 0.0% % 326Q365Y 2.0% 0.5% 0.1% 0.0% 0.1% 0.0% 0.0% %

INDUSTRIAL APPLICABILITY

The mutant GDH of the present invention has improved substratespecificity to glucose and can be suitably used for measurement ofglucose using a glucose sensor or the like.

1. An isolated mutant glucose dehydrogenase of SEQ ID NO: 3, whereinsaid mutant glucose dehydrogenase consists of a replacement of the aminoacid residue at position 365 with another amino acid residue, and/orconsists of a substitution, deletion, insertion or addition of 1 to 10amino acid residues other than the amino acid residue at position 365with another or other amino acid residue at the 324th, 326th, 333rd,334th, 368th, 369th, 376th, 377th, 418th, 419th, 436th, 433rd, 448th,472nd, 475th, 525th or 529th positions in the amino acid sequence of SEQID NO: 3, wherein said mutant glucose dehydrogenase has an increasedsubstrate specificity to glucose compared to the glucose dehydrogenaseof SEQ ID NO:3.
 2. The isolated mutant glucose dehydrogenase accordingto claim 1, consisting of a replacement of serine at position 365 withanother amino acid.
 3. The isolated mutant glucose dehydrogenaseaccording to claim 1, which has a reduced reactivity to a disaccharidecompared with the glucose dehydrogenase of SEQ ID NO:
 3. 4. The isolatedmutant glucose dehydrogenase according to claim 3, wherein thedisaccharide is maltose.
 5. The isolated mutant glucose dehydrogenaseaccording to claim 4, which has a reactivity to maltose in a degree of20% or less of reactivity to glucose.
 6. The isolated mutant glucosedehydrogenase according to claim 1, wherein the replacement amino acidresidue is an amino acid residue selected from phenylalanine, tyrosine,tryptophan and histidine residues.
 7. The isolated mutant glucosedehydrogenase according to claim 1, wherein at least one positionselected from the 326th, 472nd, 475th and 529th positions is replacedwith another or other amino acid residues.
 8. The isolated mutantglucose dehydrogenase according to claim 7, wherein the 472nd positionis replaced with another or other amino acid residues.
 9. The isolatedmutant glucose dehydrogenase according to claim 7, wherein the 475thposition is replaced with another or other amino acid residues.
 10. Theisolated mutant glucose dehydrogenase according to claim 7, wherein boththe 472nd position and the 475th position are replaced with other aminoacid residues.
 11. The isolated mutant glucose dehydrogenase accordingto claim 7, wherein the 326th position is replaced with another or otheramino acid residues.
 12. The isolated mutant glucose dehydrogenaseaccording to claim 7, wherein the 529th position is replaced withanother or other amino acid residues.
 13. The isolated mutant glucosedehydrogenase according to claim 8, wherein the amino acid residue at aposition corresponding to the 472nd position is replaced with an aminoacid residue selected from aspartic acid, glutamic acid, phenylalanine,tyrosine, isoleucine, asparagine and histidine residues.
 14. Theisolated mutant glucose dehydrogenase according to claim 9, wherein theamino acid residue at the 475th position is replaced with histidine orserine residue.
 15. The isolated mutant glucose dehydrogenase accordingto claim 11, wherein the amino acid residue of the 326th position isreplaced with glutamine or valine residue.
 16. The isolated mutantglucose dehydrogenase according to claim 12, wherein the amino acidresidue at the 529th position is replaced with tyrosine, histidine ortryptophan residue.
 17. An isolated mutant glucose dehydrogenase of theglucose dehydrogenase having the amino acid sequence of SEQ ID NO: 3,wherein said mutant glucose dehydrogenase consists of: (i) substitutionof the amino acid residue at position 365 in the amino acid sequence ofSEQ ID NO: 3, (ii) substitution of aspartic acid residue for anotheramino acid residue at position 472, and (iii) substitution of histidineresidue for another amino acid residue at position 475, wherein saidmutant glucose dehydrogenase has increased substrate specificity toglucose, compared to the glucose dehydrogenase comprising the amino acidsequence of SEQ ID NO:
 3. 18. The isolated mutant glucose dehydrogenaseaccording to claim 17, wherein the other amino acid residue or residuesare selected from phenylalanine, tyrosine and tryptophan residues. 19.An isolated FAD-binding type mutant glucose dehydrogenase comprising theamino acid sequence of SEQ ID NO:
 7. 20. An isolated mutant glucosedehydrogenase complex comprising at least the mutant glucosedehydrogenase according to claim 1 and an electron transfer subunit. 21.The isolated glucose dehydrogenase complex according to claim 20,wherein the electron transfer subunit is cytochrome C.
 22. A glucoseassay kit comprising the mutant glucose dehydrogenase according toclaim
 1. 23. A glucose sensor comprising the mutant glucosedehydrogenase according to claim
 1. 24. A glucose assay kit comprisingthe mutant glucose dehydrogenase complex according to claim
 20. 25. Aglucose sensor comprising the mutant glucose dehydrogenase complexaccording to claim 20.